The present invention relates to methods of pooling or localizing the allantoic fluid present in avian shell eggs, so that the fluid may be readily sampled, assessed, or injected with a substance. Thus, the present invention also relates to a method of directing a probe into the allantois of an egg for the purpose of sampling or examining the allantoic fluid contained therein, or to a method of injecting a substance into the allantois of a shell egg.
The allantois or allantoic sac is a portion of an avian egg that develops and then enlarges in the egg from about the 5th to 13th day, and thereafter is absorbed and diminishes in size until the egg is hatched. The allantoic sac functions as a respiratory surface, a calcium adsorbing tissue, a liquid waste receptacle for urine secreted by the mesonephros, and as a water reservoir to sustain later stage development of the embryo. The allantoic sac reaches its peak volume at about the 11th day of incubation and begins to diminish in size after about the 13th day. Because the allantoic sac essentially grows to surround the embryo, it normally exists as a relatively thin layer under the inner shell membrane which presents a difficult target for injection even during the period of maximum volume.
Methods for producing influenza and other viral vaccines involve the growth of the viruses in embryonated chicken eggs. Typically, each egg must be inoculated by injection of a small volume of virus into the allantoic cavity. The injected eggs are then incubated and opened to allow harvesting of the allantoic fluid and the virus growing therein. See, e.g. U.S. Pat. No. 4,338,296 to Lobmann.
Additionally, various materials may be injected into the avian embryo in ovo, or into the various compartments within the embryonated avian egg, to achieve beneficial effects in a subsequently hatched chick. Such: beneficial effects include increased growth, prevention of disease, increasing the percentage hatch of multiple incubated eggs, and otherwise improving physical characteristics of hatched poultry. Additionally, certain types of vaccinations are suitable for in ovo administration (see U.S. Pat. No. 4,458,630 to Sharma).
One method of sampling or injecting the allantoic fluid of an avian egg involves candling of individual eggs to locate the allantoic and then injecting or sampling the allantois by hand using a syringe. Injection into the allantois by hand has been practiced in the biological sciences for the purpose of culturing various microorganisms therein. However, because of the difficulty of reliable targeting the allantois using high volume or automated methods, the poultry industry has not generally used the allantois as a target for injection.
A number of automatic egg injection devices have been developed. These include U.S. Pat. No. 5,056,464 to Lewis; U.S. Pat. Nos. 4,903,635 and 4,681,063 to Hebrank; U.S. Pat. No. 5,136,979 to Paul et al.; and U.S. Pat. Nos. 4,040,388, 4,469,047 and 4,593,646 to Miller, which are incorporated herein by reference in their entirety.
Recently, efforts by the poultry industry to inject into the allantois using vertical downward injection through the large end of a vertically oriented egg by traversing the air sac and air sac membrane have been reported, but efforts to accomplish this on a large scale using conventional egg injection equipment have produced mixed results with a significant number of injections discharging into the air sac or the amnion instead of the allantois. It is therefore an object of the present invention to provide a method whereby the allantoic sac may be reliably targeted for injection with, for example, microorganisms that are to be cultivated in the egg or materials that are beneficial to the embryo. It is a further object of the present invention to provide a method whereby the allantoic sac may be reliably targeted for piercing with a probe such as a hypodermic needle for purposes of analyzing or sampling the allantoic fluid contained therein. It is a further object of the present invention to provide an improved method for reliably injecting into the allantois of an egg on a large scale using conventional egg injection equipment, and to provide a method that injects or reaches the allantois without piercing the air cell membrane. It is a further object of this invention to provide a method for utilizing an improved egg tray in the process of injecting into the allantois of an egg on a large scale using conventional egg equipment. These and other objects of the invention will be apparent to those skilled in the art in view of the ensuing description and claims.
In accordance with the method of the present invention, an avian shell is rotated or tilted so that the major axis of the egg is from about 10 to 180 degrees from the vertically upright position, where the upright large end of the egg containing the air sac defines the zero degree position. Such orientation of the egg away from the vertically upright position causes the allantois to pool within the egg on the uppermost side of the egg. This enlarged area of the allantoic sac is thereupon targeted for injection or sampling; the injector or sampling device need not traverse the air cell to reach the allantois.
The angle of injection into the egg may be vertically downward, horizontal or at any convenient angle. When the long axis of the ego is greater than about 10 degrees or about 20 degrees from vertical, the allantoic sac is conveniently targeted by downward vertical injection using conventional egg injection equipment; the angle of egg orientation can be chosen so that the injector or sampling device does not traverse the air cell of the egg. Injection of the egg in this manner is facilitated by the methods of the present invention utilizing egg trays designed to orient a plurality of avian eggs at a predetermined angle, used in combination with conventional egg-injection equipment using a vertical downwardly traveling injection needle.
Where the allantois of an egg is targeted for injection, the injection needle is directed into the egg at a predetermined angle and depth to target the area of the allantoic sac having the greatest volume and hence the largest target area. The relationship between egg orientation, allantoic sac configuration and location and direction of injection to optimize the probability of injection into the allantois will be apparent from the following drawings and detailed description of the invention.
A first aspect of the present invention is a method for injecting a substance into the allantois of an avian egg during the last quarter of incubation. The long axis of the egg is oriented to a predetermined angle from about 10 degrees to 180 degrees from vertical (where the large end of the egg in an upwardly vertical position defines zero degrees vertical). This orientation of the egg causes the allantois of the egg to pool and enlarge the allantoic sac in an area of the egg under the upper portion of the egg shell. A needle is inserted downwardly through the shell of the egg and directly into the enlarged allantoic sac, and the substance is injected into the allantois.
A further aspect of the present invention is a method for injecting a substance into the allantois of a plurality of chicken eggs in the 16th to 19th day of incubation. The eggs are oriented on an egg tray to a predetermined angle from about 10 degrees to 180 degrees from vertical (where zero degrees vertical is defined by the large end of the egg in a vertically upward position). This orientation of the egg causes the allantois of each egg to pool and enlarge the allantoic sac in a predetermined area of the egg under the upper portion of the egg shell. An injection needle is inserted downwardly into each egg through the shell and into the enlarged allantoic sac, and the substance is injected through the needle into the allantois of each egg.
A further aspect of the present invention is a method for directing a probe into the allantois of an avian egg in the last quarter of incubation. The long axis of the egg is oriented to a predetermined angle from about 10 degrees to 180 degrees from vertical (where the large end of the egg in an upwardly vertical position defines zero degrees vertical). This orientation causes the allantois of the egg to pool and enlarge the allantoic sac under the upper portion of the egg shell. A probe is inserted downwardly through the shell of the egg and into the enlarged allantoic sac.